Nigral innervation of cholinergic and glutamatergic cells in the rat mesopontine tegmentum: light and electron microscopic anterograde tracing and immunohistochemical studies.

The substantia nigra (SN) has long been known as an important source of afferents to the pedunculopontine tegmental nucleus (PPN). However, it has not been established which of the chemospecific cell populations receive this synaptic input. We sought to address this issue by a correlative light and electron microscopic approach that combines anterograde tracing of nigral efferents with pre-embedding choline acetyltransferase (ChAT) and/or glutamate (Glu) immunohistochemistry. Following large bilateral injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) in the SN, the labeled nigrotegmental fibers were concentrated in a small area of the mesopontine tegmentum which contained very few ChAT-immunoreactive (ChAT-ir) cell bodies. However, strands of fine varicose fibers penetrated to adjacent regions of the PPN which harbored numerous cholinergic perikarya. The anterogradely labeled boutons were often seen in the proximity of ChAT-ir perikarya and dendrites, but the majority (82-93%) established symmetric synaptic junctions with noncholinergic profiles. In the pars dissipata of the PPN (PPNd), one-third of the labeled terminals synapsed onto noncholinergic perikarya and primary dendrites, while in the pars compacta of the PPN (PPNc) axosomatic synapses were rare. The possibility that the perikarya receiving a rich synaptic input from the SN are glutamatergic was tested in experiments combining anterograde transport of biotinylated tracers biocytin and dextran-amine (BDA) with glutamate immunohistochemistry. In double-labeled sections, Glu-ir perikarya within the terminal plexus of nigrotegmental fibers were surrounded by synaptic terminals. The PPNd also contained retrogradely BDA-labeled neurons which were contacted by anterogradely labeled terminals. These results indicate that although a small subpopulation of cholinergic neurons in the mesopontine tegmentum receive direct synaptic input from the SN, the primary target of nigrotegmental fibers are glutamatergic cells in the PPNd. Our results also provide ultrastructural evidence that some nigrotegmental fibers innervate pedunculonigral neurons.

Nigrothalamic projections and nigrothalamocortical pathway to the medial agranular cortex in the rat: single- and double-labeling light and electron microscopic studies.

Although the rat medial agranular cortex (AGm) has been implicated in a variety of motor functions, the source of the afferents impinging upon thalamic neurons projecting to the AGm is not directly known. The main purpose of this study was to determine whether the AGm is a major recipient of the nigrothalamocortical pathway. This issue was addressed by two sets of experiments. First, the organization of the nigrothalamic projections was studied by light and electron microscopy following injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) into the pars reticulata of the substantia nigra (SNR). The major finding of this study was the disclosure of a heretofore unknown projection to the rostromedial part of the ventral anterior-ventral lateral complex (VAL). This projection originates exclusively from the ventral portion of the SNR and is comparable in strength to the well-known nigrothalamic projection to the ventromedial nucleus (VM). Electron microscopic examination revealed differences in the synaptic organization of nigral terminals in the VAL and the VM. A large proportion of the labeled terminals in the VAL was involved in axosomatic synapses, whereas, in the VM, the axosomatic synapses were rare, and 67% of nigral terminals were found in contact with thin dendrites. To assess a possible disynaptic nigrothalamocortical pathway to the AGm, a double-labeling strategy combining PHA-L injections in the SNR and pressure injections of the retrograde tracer, cholera toxin subunit B (CTB) in the AGm was employed. The greatest density of CTB-labeled neurons was found in the rostral and central portion of the VAL, coincident with the nigrothalamic labeling originating from the ventral SNR. Electron microscopic analysis confirmed that some of the PHA-L-labeled terminals established synaptic contacts with the CTB-labeled cell bodies and large dendrites. In conclusion, our findings indicate that there exist two different nigrothalamocortical pathways through the motor thalamus in the rat. The SNR-VAL-AGm cortical projection may play a role in oculomotor functions, whereas the SNR-VM-cortical pathway has been implicated in arousal mechanisms.

The use of peroxidase substrate Vector VIP in electron microscopic single and double antigen localization.

Very few chromogens used in immunoperoxidase reactions can be combined to simultaneously localize two neural antigens with different labels at both light (LM) and electron (EM) microscopic levels. The objective of this study was to investigate the EM properties of a novel purple chromogen introduced by LM immunostaining by Vector Laboratories under the commercial name Vector VIP. The Vector VIP (VIP) was employed to demonstrate anterogradely transported Phaseolus vulgaris-leucoagglutinin (PHA-L), retrogradely transported cholera toxin subunit B (CTB) and acetylcholine synthesizing enzyme choline acetyltransferase (ChAT) in single and double antigen immunostaining in combination with the chromogen 3,3'diaminobenzidine (DAB). The VIP reaction product proved resistant to loss during post-fixation in OSO4 and dehydration in acetone. In EM preparation, the VIP reaction product was granular in appearance and easily distinguishable from the diffuse reaction product of DAB. Compared to the chromogen benzidine dihydrochloride (BDHC), the VIP reaction procedure is much simpler, more sensitive and consistently generates the same texture of the electron-dense precipitate. This study demonstrates the usefulness of VIP as a chromogen for correlative LM and EM immunoperoxidase staining. The VIP can be used either in single or double immunostaining in combination with DAB. In addition, we have examined the EM properties of another commercial chromogen, peroxidase substrate Vector SG (SG). The blue-gray reaction product of this chromogen is strongly osmiophilic and the electron-dense precipitate appears amorphous.

Notes on a light and electron microscopic double-labeling method combining anterograde tracing with Phaseolus vulgaris leucoagglutinin and retrograde tracing with cholera toxin subunit B.

Investigations of monosynaptic connections in the central nervous system have been hindered by the lack of compatible markers that can be used at both light and electron microscopic levels. In attempts to determine synaptic contacts between fibers originating in the substantia nigra and neurons projecting to the spinal cord, we have developed a double immunolabeling technique using anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L) and retrograde transport of unconjugated cholera toxin subunit B (CTB). In this report, we describe technical modifications which consistently produced superior labeling together with adequate ultrastructural preservation of the tissue and discuss the advantages of the two tracers.

Cholinergic and non-cholinergic neurons in the rat pedunculopontine tegmental nucleus.

Choline acetyltransferase immunhistochemistry was employed at light and electron microscopic levels in order to determine the distribution of cholinergic neurons in two subdivisions of the rat pedunculopontine tegmental nucleus that were previously defined on cytoarchitectonic grounds, and to compare the synaptic inputs to cholinergic and non-cholinergic somata in the subnucleus dissipatus, which receives major input from the substantia nigra. Large cholinergic neurons were found in both the pars compacta and the pars dissipata of the pedunculopontine nucleus. However, they were intermingled with non-cholinergic neurons and did not respect the cytoarchitectural boundaries of the nucleus. Ultrastructural study showed that all cholinergic neurons in the subnucleus dissipatus exhibited similar features. The majority had large somata (largest diameter greater than or equal to 20 microns) containing abundant cytoplasmic organelles and nuclei displaying a few shallow invaginations. Synaptic terminals on the cholinergic cell bodies were scarce and unlabeled boutons containing spherical synaptic vesicles and establishing asymmetric synaptic junctions were the dominant type. In contrast, the non-cholinergic neurons presented prominent differences in the size of their somata as well as in the distribution of axosomatic synapses. Two almost equally represented classes of non-cholinergic neurons which are referred to as large (largest diameter greater than or equal to 20 microns) and small (largest diameter less than 20 microns) were recognized. Large non-cholinergic cell bodies were ultrastructurally similar to the cholinergic ones, but they received rich synaptic input by unlabeled nerve terminals which contained pleomorphic vesicles and were engaged in symmetric synaptic junctions. Small non-cholinergic cell bodies were characterized by deeply invaginated nuclei surrounded by a narrow rim of cytoplasm, and were often found near or in direct apposition to the cholinergic somata. Their major input consisted of axosomatic boutons containing round synaptic vesicles. These results demonstrate that cells in the pedunculopontine tegmental nucleus are differentiated with regard to their axosomatic synaptic inputs which may influence their firing properties. Furthermore, they support previous suggestions that nigral afferents may be preferentially distributed to a subpopulation of the pedunculopontine neurons.

Nigropedunculopontine projection in the rat: an anterograde tracing study with phaseolus vulgaris-leucoagglutinin (PHA-L).

The termination of the substantia nigra pars reticulata efferents in the nucleus tegmenti pedunculopontinus was studied in the rat by using the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L). Both large and small injections of PHA-L in various portions of the substantia nigra pars reticulata labeled varicose fibers in the ipsilateral and contralateral nucleus tegmenti pedunculopontinus, subnucleus dissipatus as well as in the ipsilateral nucleus tegmenti pedunculopontinus, subnucleus compactus. However, the bulk of the nigral fibers appeared to terminate in the medial two-thirds of the ipsilateral subnucleus dissipatus of the pedunculopontine nucleus and exhibited a discrete dorsoventral topographical pattern. The terminal plexus displayed patches of uneven density, which was partly due to the numerous fiber bundles passing through the pedunculopontine nucleus, but also to an obvious preference of nigral fibers for some cells. Electron microscopic examination confirmed that nearly all of the varicosities observed in the light microscope contained synaptic vesicles and represented either terminal boutons or boutons en passant. The labeled boutons were elongated (average length: 1.5 microns) and consistently contained a prominent group of mitochondria. The results suggest that the nigral input to the nucleus tegmenti pedunculopontinus may be directed toward specific subpopulation(s) of pedunculopontine neurons and may influence not only cells in the subnucleus dissipatus, but also in the subnucleus compactus.

Descending brainstem projections of the pedunculopontine tegmental nucleus in the rat.

Descending brainstem projections from the pedunculopontine tegmental nucleus (PPN) were studied in the rat by use of the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) and the retrograde tracer lectin-conjugated horseradish peroxidase (HRP-WGA). Results of these experiments demonstrated prominent bilateral projections to the pontomedullary reticular nuclei, but direct connections to the motor and sensory nuclei of the cranial nerves could not be ascertained. The PPN fibers terminated mainly in the pontine reticular nuclei oralis and caudalis and in ventromedial portions (pars alpha and pars ventralis) of the gigantocellular reticular nucleus. A smaller number of labeled fibers distributed to more dorsal regions of the gigantocellular nucleus, lateral para-gigantocellular, ventral reticular nucleus of the medulla and lateral reticular nucleus. Although a significant number of PHA-L labeled fibers was seen in two cases in the contralateral medial portion of the facial nucleus, and all cases exhibited a sparse predominantly ipsilateral projection to the lateral facial motor neurons, the retrograde tracing experiments have revealed that these facial afferents originated in the nuclei surrounding the PPN. The results are discussed in the context of PPN involvement in motor functions. It is suggested that the PPN may participate in a complex network involved in the orienting reflex.

Origin of ascending and spinal pathways from the nucleus tegmenti pedunculopontinus in the rat.

The distribution and collateralization of ascending and descending projections from neurons in the nucleus tegmenti pedunculopontinus (PPN) were studied in the rat by using retrograde transport of HRP, HRP/WGA, and fluorescent dyes. The PPN and its two subdivisions, the subnucleus compactus (PPNc) and subnucleus dissipatus (PPNd), were delineated on sagittal Nissl-stained sections by using cytoarchitectural features as guidelines. Large bilateral pressure injections of HRP and/or fluorescent dyes into the cervical cord retrogradely labeled moderate numbers of fusiform and polygonal PPN cells which ranged in size between 65 and 390 microns2. The labeled cells were scattered throughout the PPNd and were somewhat more numerous in the medial half of the subnucleus. The PPNc contained only occasional labeled cells in its ventralmost portion. Following single unilateral HRP/WGA injections in the striatum, globus pallidus, entopeduncular nucleus, subthalamus, or the substantia nigra, the distribution of the labeled cells was similar to that of the spinal cord-projecting PPN neurons. Multiple HRP injections were then made bilaterally in the substantia nigra and the entopeduncular nucleus and/or subthalamus in order to label the entire population of PPN neurons projecting to the basal ganglia. In this case, not only the PPNd but also the PPNc contained a substantial number of retrogradely labeled cells. The rostrally projecting PPN cells outnumbered 5.4 times those projecting to the spinal cord, and their somata were somewhat larger, ranging between 114 and 472 microns2. While both fusiform and polygonal shapes were encountered, the polygonal cell somata were more numerous. In the double-labeling experiments, Granular Blue and Diamidino Yellow Dihydrochloride were injected into the cervical cord and the entopeduncular nucleus or subthalamus. In general, these experiments confirmed the extensive overlap of forebrain- and spinal cord-projecting neurons within the PPNd and the quantitative preponderance of ascending neurons. They also demonstrated that these two projection systems originate largely from separate cell populations since the double-labeled cells always composed less than 5% of the labeled neurons. The results confirm the existence of a direct PPN projection to the spinal cord. This pathway originates mainly in the PPNd and appears to be quantitatively weaker than the PPN projections to the forebrain. The spinal cord-projecting cells are not spatially segregated from the cells projecting to the basal ganglia, but they represent a separate population of the PPN projection neurons.

Morphology of the substantia nigra pars reticulata projection neurons intracellularly labeled with HRP.

The technique of intracellular recording and staining of the same neuron with horseradish peroxidase (HRP) was used to study the soma-dendritic and axonal morphology of nigrothalamic and nigrotectal cells in the rats. The nigrothalamic and nigrotectal cells were spread throughout the dorsoventral extent of the pars reticulata (SNR) and exhibited the same soma-dendritic and axonal features. Both populations consisted of medium-sized and large cells with extensive dendritic fields overlapping in all three directions. Their axons collateralized within the substantia nigra (SN) and in the mesencephalic tegmentum. The intrinsic collaterals were thin and branched partly within the dendritic field of a parent cell partly in remote regions of the SNR, and even in the pars compacta (SNC). The extrinsic branches involved thin arborizations in the rostroventral mesencephalic reticular substance and thicker descending and ascending collaterals. This material was supplemented by physiologically nonidentified HRP stained medium-sized and large neurons located in the SNR. The two kinds displayed the same extent and orientation of their dendrites but the branching patterns differed slightly. Proximal dendrites of all cells were coarse and smooth; thinner distal dendrites had varicosities and spinelike appendages. Some dendrites, specially those near the crus cerebri, terminated in dendritic thickets bearing many pleomorphic appendages. The orientation of dendritic fields varied with dorsoventral position of cells within the SNR. The most ventral region of the SNR contained neurons with dendrites oriented parallel to the crus cerebri and thus remained confined to the deepest stratum. The dendrites of cells in the central region of SNR were oriented mainly anteroposteriorly and ventrally, the ventral dendrites terminating in the ventralmost layer. Cells in the dorsolateral part of the SNR were characterized by the large dorsoventral extent of their dendrites which penetrated the entire thickness of SN. This variation in the arrangement of dendritic fields indicates that the SN is organized in three dorsoventral layers.

Neuronal interactions in the substantia nigra pars reticulata through axon collaterals of the projection neurons. An electrophysiological and morphological study.

Substantia nigra pars reticulata (SNr) neurons, antidromically activated following stimulation of the dorsal thalamus and/or superior colliculus were intracellularly stained with HRP. Light microscopic analysis revealed that the labeled SNr neurons have axon collaterals arborizing within SNr. Axon collaterals of SNr neurons partially overlapped with the dendritic fields of their parent cells and also extended beyond the parent dendritic fields. The labeled axon terminals did not closely appose the parent cell processes, suggesting that the collaterals most likely terminate on neurons other than the parent cell. Electrical stimulation of either the thalamus or the superior colliculus induced monosynaptic and polysynaptic IPSPs in SNr cells. The polysynaptic IPSPs evoked from thalamic stimulation disappeared following hemitransection of the brain just rostral to the thalamus while the monosynaptic IPSPs remained the same. Since there are no known afferents from either thalamus or superior colliculus to SNr, we consider that these monosynaptic IPSPs are due to activation of the recurrent collaterals of SNr projection cells. The results of this study indicate that projection neurons of SNr also have an inhibitory role within the SNr.

Morphological changes in rat brain induced by L-cysteine injection in newborn animals.

A single subcutaneous injection of L-cysteine (1.2 mg/g body weight) to rats 4 days after birth was followed by atrophy of the brain which was well developed 27--32 days after the injection. It was apparent that the lesioned animals could be divided into two groups (type 1 and 2) on account of the degree of brain atrophy. In type 1, which was observed in 80% of the animals, the body weight was unchanged, but the total brain weight was reduced by about 20%. The brain structures most affected were cerebral cortex, hippocampus and thalamus, each having a 30--40% reduction in wet weight. The atrophy of the posterior part of cortex was particularly pronounced in this type of lesion. In type 2 lesion, which appeared in 10% of the survivors, the atrophy was much more severe. There was a 50% reduction in wet weight of brain and in body weight. The most prominent finding was the atrophy of the whole cortex and the hippocampus which were reduced by 80 and 60% of wet weights respectively. In this type of lesion significant morphological changes were observed in several brain regions such as caudato-putamen, thalamus, pons, medulla oblongata, spinal cord and cerebellum.

[Somatotopic organization of the vestibulospinal tract in the toad]. / Organizzazione somatotopica della via vestibolospinale nel rospo.

The origin of the vestibulospinal projection in the toad has been investigated by using the method of the retrograde axonal transport of HRP injected at various levels of the spinal cord. The vestibulospinal projection, in this species, was found to be somatotopically organized, since neurons projecting to the cervical segments of the spinal cord were located within the rostromedial part of the ventral vestibular nucleus and those neurons projecting to the lumbosacral segments of the spinal cord were located within the caudolateral part of that nucleus. This pattern of organization of the vestibulospinal projection in amphibia is similar to that described in mammals and birds.

Mesencephalic and diencephalic afferents to the superior colliculus and periaqueductal gray substance demonstrated by retrograde axonal transport of horseradish peroxidase in the cat.

The mesencephalic and diencephalic afferent connections to the superior colliculus and the central gray substance in the cat were examined by means of the retrograde transport of horseradish peroxidase (HRP). After deep collicular injections numerous labeled cells were consistently found in the parabigeminal nucleus, the mesencephalic reticular formation, substantia nigra pars reticulata, the nucleus of posterior commissure, the pretectal area, zona incerta, and the ventral nucleus of the lateral geniculate body. A smaller number of cells was found in the inferior colluculus, the nucleus of the lateral lemniscus, the central gray substance, nucleus reticularis thalami, the anterior hypothalamic area, and, in some cases, in the contralateral superior colliculus, Forel's field, and the ventromedial hypothalamic nucleus. Only the parabigeminal nucleus and the pretectal area showed labeled cells following injections in the superficial layers of the superior colliculus. In the cats submitted to injections in the central gray substance, labeled cells were consistently found in the contralateral superior colliculus, the mesencephalic reticular formation, substantia nigra parts reticulata, zona incerta and various hypothalamic areas, especially the ventromedial nucleus. In some cases, HRP-positive cells were seen in the nucleus of posterior commissure, the pretectal area, Forel's field, and nucleus reticularis thalami. A large injection in the mediodorsal part of the caudal mesencephalic reticular formation, which included the superior colliculus and the central gray substance, resulted in numerous labeled cells in nucleus reticularis thalami. The findings are discussed with respect to the suggested functional division of the superior colliculus into deep and superficial layers. Furthermore, the possible implications of labeled cells in zona incerta and the reticular thalamic nucleus are briefly discussed.

A study on the postulated transoptic regulation of the intraocular pressure.

The possibility that fibers regulating the intraocular pressure originate in the hypothalamus and course in the optic nerve, has been investigated in albino rabbits with different techniques. By means of horseradish perixodase tracing-technique, oculohypothalamic but no hypothalamoocular connections were observed. The water-loading effect on the intraocular pressure was studied after unilateral optic nerve transection. The transection was performed in three experimental groups in the following way: Intracranial transection of one optic nerve, retrobulbar transection of one optic nerve and sham operation on the other side, retrobulbar transection of one optic nerve without sham operation on the other side. In contrast to previous reports, we found no significant side difference in intraocular pressure after water-loading in any of these groups.